The invention relates to methods and reagents for determining iron binding capacity in a sample, e.g., total iron binding capacity (TIBC) in a sample.
In order to assess a patient""s status in terms of iron-deficiency anemia, it is common practice to measure two blood parameters: (1) serum Fe concentration and (2) serum TIBC.
Since iron exists in the blood only bound to proteins (primarily transferrin and ferritin), it is useful to know the total amount of (bound) iron plus the maximum potential for a patient to bind additional iron which may be given as a supplement to people who have iron deficiency.
Total iron (Fe) is commonly measured simply by lowering the pH to release all the iron from the binding proteins and then allowing the ionized iron, the Fe++ and Fe+++ forms, to react with a dye, e.g. ferene or ferrozine, which forms a colored complex that is directly related to the serum iron concentration.
TIBC is an additional measurement which measures the total capacity of the sample to bind ironxe2x80x94this number includes any sites which are bound to iron as well as to sites which are not occupied by iron but which could be so occupied. Typically only about one third of the possible iron binding sites in serum are saturated under normal conditions.
Accordingly, in one aspect, the invention features, a method of evaluating a sample, e.g., to determine TIBC. The method includes:
providing a sample;
contacting the sample (under conditions wherein iron binds to the iron binding sites in the sample) with a sufficient amount of iron such that the iron binding sites (e.g., iron binding sites on serum proteins) in the sample are saturated with iron;
sequestering the free iron in the sample to form a TIBC sample phase (e.g., a supernatant) which does not include the sequestered free iron;
evaluating the amount of concentration of iron in the TIBC sample phase, thereby of evaluating a sample, e.g., to determine TIBC
In another aspect, the invention features, a method of evaluating a sample, e.g., to determine TIBC. The method includes:
providing a sample, e.g., a serum sample;
contacting the sample (under conditions wherein iron binds to the iron binding sites in the sample) with a sufficient amount of iron such that the iron binding sites (e.g., iron binding sites on serum proteins) in the sample are saturated with iron;
contacting the sample with a magnetically responsive particle (MRP) which includes an iron binding ligand;
applying a magnetic field to the sample, thereby forming a TIBC sample phase, e.g., a supernatant, which does not include the MRP;
evaluating the amount of concentration of iron in the TIBC sample phase, thereby of evaluating a sample to determine TIBC
In another embodiment the high iron concentration solution is added to the sample, allowed time to saturate all the available sites. After this step the MRP""s are added.
In a preferred embodiment the MRP""s and high iron concentration solution are mixed, and used as a single reagent. In this embodiment the affinity of iron for the sites in the sample, e.g., serum protein sites, is much greater than the affinity of iron for the solid-phase ligand.
In another embodiment the MRP""s and the sample are first combined, then the high Fe concentration solution is added.
In another aspect, the invention includes, a preparation of a reagent disclosed herein.
In another aspect, the invention includes, a reaction mixture disclosed herein, e.g., a reaction mixture which includes a sample and one or more reagents disclosed herein.
Methods, reagents, and reaction mixtures of the invention provide advantageous methods of determining TIBC. TIBC methods of the invention can (a) eliminate centrifugation, (b) be rapid, (c) use small amounts of serum (e.g., less than 500 uL), and (d) can be adapted to on-line automation or semi-automation.
All publications, patent applications, and patents cited herein are incorporated by reference. Other features and advantages of the invention will be apparent from the following description and from the claims.